B. Protein Blotting

A general protocol for sample preparation.

Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.

Western Blot Reprobing Protocol

Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.

(Optional) To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST. Incubate membrane with LumiGLO® with gentle agitation for 1 min at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film.

Wash membrane again four times for 5 min each in TBST.

The membrane is now ready to reuse. Start detection at the "Membrane Blocking and Antibody Incubations" step in the Western Immunoblotting Protocol.

Source / Purification

Background

Telomeric repeat-binding factor 2-interacting protein (TERF2IP, also known as RAP1) is a component of the Shelterin Complex, a multi-protein complex that binds and organizes telomeres into T-loop structures to prevent them from being recognized by the cell as DNA double stranded breaks (1,2). The Shelterin Complex is composed of TERF2IP, TIN2 and TPP2 proteins, in addition to three DNA binding proteins that function to recruit the complex to telomeres: TRF1 and TRF2 bind double-stranded TTAGGG repeats found at telomeres, while the POT1 protein binds single-stranded TTAGGG repeats found at the very end of the telomeres (2). Together, these proteins function to protect telomeres and ensure proper replication and processing of chromosome ends. Recent studies have shown that TERF2IP is dispensable for maintenance of telomere length, organization of telomeric chromatin, and regulation of telomeric transcription (3,4). However, TERF2IP is required for inhibition of homology-directed repair (HDR), which can create undesirable telomeric sister chromatid exchange (3,4). In addition to its role in telomere maintenance, TERF2IP is also found in the cytoplasm, where it functions as an IκB kinase (IKK) adaptor protein and regulates NF-κB-dependent gene expression (5). TERF2IP forms a complex with IKKs and is critical for proper recruitment of IKKs to and activation of the p65 subunit of NF-κB. Elevated levels of TERF2IP have been found in breast cancer cells with NF-κB hyperactivity, and knockdown of TERF2IP sensitizes these cells to apoptosis, further identifying TERF2IP as a potential cancer therapeutic target (5).